Ustekinumab is a fully human IgG1 monoclonal antibody targeting interleukin (IL)-12/23
Ustekinumab is a fully human IgG1 monoclonal antibody targeting interleukin (IL)-12/23 p40 subunit. on CD4+ GW842166X T cell function have not been fully investigated so far. In this study, we explored changes in cytokine production by memory CD4+ T cells as well as in the differentiation of na?ve T cells to helper T cell (Th) 1, Th2, or Th17 cells in psoriasis patients treated with ustekinumab. The effect of the treatment on T cell receptor repertoire diversity was also evaluated. The results showed that ustekinumab improves clinical manifestation in patients with psoriasis without affecting cytokine production in memory T cells, T cell maturation, or T cell receptor repertoire diversity. Although the number of patients is limited, the present study suggests that T cell immune response remains unaffected in psoriasis patients treated with ustekinumab. Introduction Psoriasis is a chronic immune-mediated skin disorder with frequent clinical relapse . The majority of patients with moderate-to-severe psoriasis require specific topical and systemic therapies including phototherapy (psoralen ultraviolet A therapy (PUVA) or narrow-band ultraviolet B (NB-UVB)), methotrexate , cyclosporine , and retinoids . However, long-term follow-up during these therapies is generally difficult because of cytotoxicity-related adverse effects, treatment failure, or patient dissatisfaction , . Recently, several biologic agents (biologics) have been reported for the treatment of psoriasis C. Biologics have high target specificity and their use is associated with limited organ toxicity. However, the risk of cancer or infection during long-term use in patients with psoriasis has not been as yet investigated. IL-12 and IL-23 play important roles in the pathogenesis of psoriasis . In psoriasis patients, IL-12 and IL-23 are involved in immune response mediated by helper Th1  and Th17 , . IL-12 and IL-23 are heterodimers with a common p40 subunit. The binding of the subunits to their respective receptors activates specific intracellular signaling pathways , . Ustekinumab (Stelara?; Janssen Biotech, Inc., Horsham, PA), a fully human IgG1 monoclonal antibody, binds to the common p40 subunit of GW842166X IL-12 and IL-23, and blocks activation of the receptors of these cytokines in dendritic cells and monocytes. Recent studies have shown significant effectiveness and safety of ustekinumab in moderate-to-severe plaque-type psoriasis during phase 2  and phase 3 clinical trials C. However, IL-12 is known to have anti-cancer activity by promoting IFN- production, therefore there is risk of cancer development due to immunosuppression. The effects of ustekinumab on the production of IL-12/IL-23 are known but its effects on T cell function are not completely understood. In the present study, we investigated the influence of ustekinumab on T cell cytokine production, differentiation of na?ve T cells and on the T cell receptor repertoire diversity in psoriasis patients. Materials and Methods Subjects Five psoriasis patients and five healthy volunteers were enrolled in this study. Patients with psoriasis eligible for the use of biologics were included in the study. Briefly, they fulfilled the rule of 10: Psoriasis GW842166X Area and Severity Index (PASI)R10, and/or Body Surface Area (BSA)R10%, and/or Dermatology Life Quality Index (DLQI)R10. The phonotypical character and response to the biologics are shown in table 1. Table 1 Background of five patients and five healthy controls. Psoriasis Treatment Protocol and Blood Sampling Schedule Ustekinumab was administrated on weeks 0, 4, and 12. In principle, ustekinumab at a dose of 45 mg was administered intradermally during each therapy. Blood was sampled one month after the third administration after obtaining written informed consent from the subjects. Blood sampling was performed three times in two psoriasis patients and in one healthy volunteer (before the first administration, and one month after the second and third administration). The investigational protocol was approved by the Institutional Review Board (IRB) of Mie University Hospital (Permit Number 2096). Antibodies and Reagents Phytohemagglutinin (PHA), Phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Purified anti-human CD3 mAb, anti-hCD28 mAb, anti-hCD8a-FITC mAb, anti-hTCR /-FITC mAb, anti-hIFN–PerCP mAb, anti-hIL-4-PerCP mAb, anti-hIL-17-PerCP mAb, anti-hTNF–PerCP mAb, and brefeldin A were purchased from BioLegend (San Diego, CA, USA). Anti-hCD4-FITC mAb, anti-hCD45RA-FITC mAb, anti-hCD3-PerCp mAb, and anti-hCD45RO-PE mAb were purchased from BD/PharMingen (San Diego, CA, USA). Foxp3-PECy5 mAb, and anti-hCD127-FITC mAb were from eBioscience (San Diego, CA, USA), and FITC/PE-human TCR BV antibodies were from Beckman Coulter (Brea, CA, USA). Anti-hIL-4 mAb, anti-hIL-12 mAb, anti- hIFN- mAb, and rhIL-12 were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant hIL-1, rhTGF-, rhIL-6, Rabbit Polyclonal to A26C2/3 and rhIL-2 were purchased from PeproTech (Princeton, NJ, USA). Complete RPMI 1640 medium was made with 10% heat-inactivated fetal bovine serum (FBS, HyClone Laboratories, INC., South Logan, UT, USA), 2.0 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Nacalai tesque, Kyoto, JAPAN). Purification of CD4+T Cells PBMCs were isolated and prepared as previously described . Briefly, PBMCs were purified from heparinized peripheral.